Soft X-ray microscopy is an imaging technique for high throughput analyses of whole, hydrated cells up to 10 micron thick. The short wavelength of X-rays used (2.4 nm) makes it possible to achieve resolution in optical sections approaching 25 nm. Since organic material absorbs approximately an order of magnitude more strongly than water at this energy, biological structures can be visualized using this natural contrast mechanism alone or in combination with one of many labeling techniques. Using tomographic reconstruction technology 3-D reconstructions can be generated and the information, which is based on X-ray absorption coefficients, is quantifiable. Immunolocalization of specific proteins can be conducted using X-ray-dense labels such as gold, silver, and transition metals. At the present time, collection of a single high-resolution tomographic data set from an entire cell requires less than 15 minutes; this time will be decreased upon completion of our automated cryotilt stage that is used for data collection (in progress). We believe that soft X-ray microscopy is a technique that can bridge the gap between light and electron microscopy and provide high-throughput quantitative cellular 3-D imaging in a way never before possible. We propose to test this hypothesis by examining some of well-characterized organisms. We will: 1) Demonstrate the ability of soft X-ray microscopy to generate 3-D structural information about the organization of yeast and alterations of that structure due to specific mutations; 2) Examine the three-dimensional distribution of specific proteins in yeast; 3) Develop additional labeling protocols to enable 3-D protein co-localizations; 4) Examine other cells in suspension, specifically T cells; and 5) Extend these studies to adherent cells. These developments will make X-ray microscopy, which rapidly generates information from whole hydrated cells at better than 50 nm resolution, an important new tool for analysis of the vast amounts of molecular information being produced on a daily basis.